Expression of COX-2,iNOS,p53 and Ki-67 in gastric mucosa-associated lymphoid tissue lymphoma

AIM: To assess the expression of cyclooxygenase-2 (COX-2),nitric oxide synthase (iNOS), p53 and Ki-67 in gastric mucosaassociated lymphoid tissue (MALT) lymphoma and clarify the relationship between COX-2 expression and iNOS or p53 expression in these patients.METHODS: The expressions of COX-2, iNOS, p53 and Ki-67 were detected in 32 gastric MALT lymphoma specimens and 10 adjacent mucosal specimens by immunohistochemical Envision method.RESULTS: COX-2 and iNOS expressions were significantly higher in gastric MALT lymphoma tissues than those in adjacent normal tissues. The expression of COX-2 was observed in 22 of 32 cases of MALT lymphoma tissues (68.8%). A positive cytoplasmic immunoreactivity for iNOS was detected in 17 of 31 cases (53.1%). COX-2 expression in gastric MALT lymphoma tissues was positively correlated with iNOS expression (r=-0.448, P=0.010) and cell proliferative activity analyzed by Ki-67 labeling index (r=0.410, P=0.020).The expression of COX-2 protein did not correlate with age,sex, stage of disease, lymph node metastasis or differentiation.The accumulation of p53 nuclear phosphoprotein was detected in 19(59.4%) of tumors, p53 protein was expressed in 11 of 23 assessed LG tumors and in 8 of 9 assessed HG tumors.The difference of p53 positivity was found statistically significant between LG and HG cases (P=0.0302). The p53 accumulation correlated with advanced clinical stage (stage Ⅲ+Ⅳ vs stage Ⅰ+Ⅱ, P=0.017). There was a significant positive correlation between COX-2 expression and p53 accumulation status (r=0.403,/=0.022). The mean PI of Ki-67 in each grade group were 36.0±7.73% in HG and 27.4±9.21% in LG. High-proliferation rate correlated with HG tumors (r=0.419, P=0.017). The correlation coefficient showed a significant positive correlation between PI and COX-2 expression in MALT lymphoma patients (r=-0.410,P=0.020).CONCLUSION: COX-2 expresses in the majority of gastric MALT lymphoma tissues and correlates with cellular proliferation and iNOS expression. COX-2 overexpression is closely associated with p53 accumulation status, iNOS and COX-2 may play a synergistic role in the pathogenesis of gastric MALT lymphoma.

15d-PGJ_2 inhibits cell growth and induces apoptosis of MCG-803 human gastric cancer cell line

AIM: To investigate the influence of peroxisome proliferator activated receptor γ (PPARγ) ligand, 15-deoxy-△12, 14-prostaglandin J2 (15dPGJ2) on the proliferation and apoptosis of MCG-803 human gastric cancer cell lines.METHODS: Cell proliferation was measured by 3H-TdR assay. Apoptosis was determined by ELISA and TUNEL staining. Protein and mRNA level of bcl-2 family and COXs were measured by Western blotting and Northern blotting respectively. PGE2 production was examined by RIA.RESULTS: 15dPGJ2 inhibited cell growth and induced apoptosis of MlCG-803 cells. The COX-2 and bcl-2/bax ratios were decreased following 15dPGJ2 treatment. The PGE2production in supernatants was also decreased. These changes were in a dose-dependent manner.CONCLUSION: 15dPGJ2 may be a useful therapeutic agent for the treatment of gastric cancer.

Expression of p57^(kip2) and its relationship with clinicopathology, PCNA and p53 in primary hepatocellular carcinoma

AIM: To investigate the expression of p57kip2 and its relationship with clinicopathology, PCNA and p53 in primary hepatocellular carcinoma (HCC). METHODS: Expression of p57kip2, PCNA and p53 in tumor tissues from 32 patients with HCC and 10 liver tissues of normal persons was detected with Elivision immunohistochemical technique. RESULTS: The p57kip2 protein positive-expression rate in HCC was 56.25%, lower than that in normal tissues (100%, P<0.05). The reduced expression of p57kip2 protein correlated significantly with moderate or low differentiation of tumor cells (P = 0.007 <0.05), high clinical stage (P= 0.041 <0.05) and poor prognosis (P= 0.036 <0.05), but did not correlate significantly with metastasis, tumor size, level of AFP and age (P>0.05). The PCNA positive-expression rate was 56.25%, which was correlated significantly with the expression of p57kip2 (P= 0.025<0.05). The p53 positive-expression rate was 46.88%, which was not correlated significantly with the expression of p57kip2 (P>0.05). CONCLUSION: There is a marked loss or absence of p57kip2 expression and high expression of PCNA in HCC, which are involved in carcinogenesis and development of HCC. The p57kip2 and p53 may induce apoptosis via different mechanisms.

Effects of AZT and RNA-protein complex (FA-2-b-β) extracted from Liang Jin mushroom on apoptosis of gastric cancer cells

To investigate the synergistic effects of 3′-azido-3′- deoxythymidine （AZT） and FA-2-b-β extracted from Ling Jin mushroom on apoptosis of gastric cancer cells MKN45 in vitro. METHODS： Ml-I- analysis was made to examine the inhibition rate of MKN45 cells treated with AZT （2.5, 5, 10 and 20 mg/L） and FA-2-b-13 （5, 10, 20 and 40 mg/L） singly and combinatively for 24, 48 and 72 h. Apoptotic effects were evaluated by morphological methods, DNA agarose gel electrophoresis and flow cytometry, respectively. Telomerase activity was estimated by TRAP- ELISA. The mRNA expression of caspase-3 and Bcl-2 were detected by RT-PCR. RESULTS： AZT and FA-2-b-13 could significantly inhibit MKN45 cell proliferation and induce its apoptosis. MKN45 cells were inhibited in dose- and time- dependent manner. The inhibition effect of AZT combined with FA-2- b-β was obviously better than that used singly （0.469 ＋ 0.022 vs 1.075 4- 0.055, P 〈 0.05, 0.325 4- 0.029 vs 0.469 ＋ 0.022 P 〈 0.01）. AZT used singly and combination of FA-2-b-β could decrease the activity of tumor cell telomerase, and AZT has synergistic function with FA- 2-b-β. A certain concentration of AZT could up-regulate the expression of caspase-3 mRNA （r = 0.9969, P 〈 0.01）, which was positively related to apoptosis rate, and could down-regulate the expression of Bcl-2 mRNA, which was negatively related to apoptosis rate （r = 0.926, P 〈 0.01）. Furthermore, the effect of AZT combined with FA-2-b-13 was significantly higher than that used singly. CONCLUSION： Combination of AZT and FA-2-b-β has an obviously synergetic effect in the gastric cancer cells MKN45, which has provided a new approach to the treatment of gastric cancer clinically.

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.

Proteomics to display tissue repair opposing injury response to LPS-induced liver injury

AIM: To examine the protein expression alterations in liver injury/repair network regulation as a response to gut-derived lipopolysaccharide (LPS) treatment, in order to anticipate the possible signal molecules or biomarkers in signaling LPS-related liver injury.METHODS: Male BALB/c mice were treated with intraperitoneal (i.p.) LPS (4 mg/kg) and sacrificed at 0, 6, 24 and 30 h to obtain livers. The livers were stained with hematoxylin and eosin for histopathologic analyses. Total liver protein was separated by two-dimensional gel electrophoresis (2-DE). The peptide mass of liver injury or repair related proteins were drawn up and the protein database was searched to identify the proteins.RESULTS: Observations were as follows: (1) TRAIL-R2 was down regulated in livers of LPS-treated mice. TNFAIP1 was significantly up regulated at 6 h, then down- regulated at 24, 30 h with silent expression during senescent stage.(2) The amount of metaxin 2 and mitochondria import inner membrane translocase subunit TIM8a (TIMM8A) was increased upon treatment with LPS, (3) P34 cdc2 kinase was significantly up-regulated 30 h after LPS administration with silent expression during senescent, 6, 24 h treated stage. (4) The amount of proteasome activator 28 alpha subunit (PA28), magnesium dependent protein phosphatase(MDPP) and lysophospholipase 2 was decreased 6 h after LPS treatment but recovered or up-regulated 24 and 30 h after LPS treatment.CONCLUSION: LPS-treated mouse liver displaying a timedependent liver injury can result in expression change of some liver injury or repair related proteins.

Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype

AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901)and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism,and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells.METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay.RESULTS: Telomerase activity was detected in well,moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively).Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 μmol/L, and was entirely inhibited at 10 μmol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P＜0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901gastric cancer cell lines (the inhibition ratio was 40.89%and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P＜0.05).Apoptosis of SGC-7901 and MKN-45 cells was detected not only by morphology and TUNEL assay but also by flow cytometry. The apoptotic rate reached 33.56% for SGC-7901 cells and 44.75% for MKN-45 cells.CONCLUSION: The viability and proliferation of gastric cancer cells can be inhibited by antisense telomerase oligomers. The growth inhibition of gastric cancer cells is correlated with concentration, time and sequence specialty of antisense oligomers. The inhibition mechanism of antisense human telomerase oligomers depends not only on the sequence specialty but also on the biological characteristics of gastric cancer cell lines.

A novel,rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+monocytes within 48 hours of in vitro culture

AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6) and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h of in vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h of in vitro culture (FastDC).METHODS: HCCLM3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/L heat inactivated human AB serum, 2 mmol/1 L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1 μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/1 polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritornas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence-activated cell sorting (FACS).Novel dendritomas were nucleus-stained with Hoechst 33258 and analyzed by confocal laser scanning microscopy.RESULTS: Mature FastDCs with highly expressed surface markers CD80, CD86, CD83 and HLA-DR were generated within 48 h in vitro. Novel dendritomas with dual red-green fluorescence were constructed fast and successfully, and FACS analysis showed that the fusion efficiency was 24.27% and the novel dendritomas expressed the same activation markers as FastDCs. Confocal laser scanning microscopy analysis showed representative images of dendritomas.CONCLUSION: Dendritomas can be formed fast with mature FastDCs from healthy human peripheral blood monocytes (PBMC) by incubation with GM-CSF and IL-4 for 24 h and by activation with proinflammatory mediators for an additional period of 24 h. Owing to shorter time required for in vitro DCs development, the generation of these novel dendritomas reduced labor and cost. This rapid method for formation of dendritomas may represent a new strategy for immunotherapy of hepatocellular carcinoma.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

EFFECTS OF ANTICOAGULATION PROTEIN DEFECT IN MATERNAL PLASMA ON SPONTANEOUS ABORTION

Objective To investigate the mechanism of anticoagulation protein defect in the pathogenesis of unexplained recurrent miscarriage. Methods Fifty-seven patients with a history of unexplained abortion were enrolled as the investigation group for tests of protein C, protein S, antithrombinⅢ(AT-Ⅲ), as well as activated protein C resistance (APC-R). The control group con-sisted of fifty healthy women with a history of normal pregnancy and delivery. Blood samples were obtained for measuring serum activity of protein C, protein S, AT-Ⅲ, and APC-R. Patients with positive APC-R were tested for factorⅤ(FⅤ) Lei-den gene mutation by PCR-RFLP method. Results Of the 57 patients, 12 (21.1%), 1 (1.8%), and 5 (8.8%) cases were found with protein S, protein C, and AT-Ⅲdeficiency respectively, and 13 (22.8%) cases with positive results of APC-R. Of the control group, no protein C or AT-Ⅲdeficiency was ever found, whereas 2 (4.0%) volunteers were presented with protein S deficiency and 3 (6.0%) with positive results of APC-R. No FⅤLeiden gene mutation was identified in all the patients with positive APC-R results. Late spontan-eous abortion cases had higher incidence of anticoagulation protein defect than the early cases. Conclusion Anticoagulation protein defect may play a role in the pathogenesis of fetal loss, especially for those occurr-ing in late stage of pregnancy.

Effects of STI571 and p27 gene clone on proliferation and apoptosis of K562 cells

AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line.p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot.MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index.RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined.The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58,P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell).CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.

Regulative Function of Telomerase and Extracelluar Regulated Protein Kinases to Leukemic Cell Apoptosis

In order to investigate the regulative function of telom erase and phosphorylated (acti- vated) extracelluar regulated protein kinase (ERK) 1and 2 in the leukemic cell lines HL - 6 0 and K5 6 2 proliferation inhibition and apoptosis,three chemotherapeutic drugs Harringtonine(HRT) , Vincristine(VCR) and Etoposide(Vp16 ) were selected as inducers.The proliferation inhibition rate was detected by MTT m ethod,the cell cycle and cell apoptosis was analyzed by flow cytometry and the telom erase activity was detected by the telom eric repeat am plification protocol(TRAP) assay and bioluminescence analysis method.The phosphorylated ERK 1/ 2 protein expression was detected by western blot method.The results showed that HRT,VCR and Vp16 could inhibit cell proliferation,induce apoptosis,inhibit telomerase activity and down- regulate the protein expres- sion of phosphorylated ERK.Itwas suggested that ERK signal transduction pathway was involved in the down- regulation of telomerase activity and the onset of apoptosis in the leukem ic cells treat- ed by HRT,VCR and Vp16 .

Rituximab in combination with cyclophosphamide, vincristine, doxorubicin and prednisone for treatment of initially diagnosed diffuse large B cell lymphoma： a multi-center clinical study

AIM: To evaluate the efficacy of rituximab combined with cyclophosphamide, vincristine, doxorubicin, and prednisone (CHOP) in treating the initially diagnosed diffuse large B cell lymphoma (DLBL). METHODS: From Apr.2002 to Feb. 2003, 52 patients were enrolled in this study. Chemotherapy was conducted with cyclophosphamide 600 mg·m^-2, vincristine 1.4 mg·m^-2,doorubicin 25mg·m^-2 on d 1 and prednisone 60 mg·d^-l for successive 5 d (standard CHOP). There were 6 courses, 3 wk each. Rituximab 375 mg·m^-2 was infused once a week, 2 d before the first course of chemotherapy (successive infusion) for 4 times on standard dose or for 6 times on extended dose. Or rituximab was infused once every 3 wk, 2 d before each CHOP (separated infusion) for 4 times on the schedule of standard dose or for 6 times on the extended dose. RESULTS: The complete response (CR) rate (60%) and total effective (100%) were achieved in 50 patients who were evaluated for efficacy, respectively. And among 34 patients in Ann Arbor stage Ⅲ and Ⅳ, 15 patients were completely relieved. The complete effective rate was 44%. Fifty patients were followed-up for (8±s 5) wk, 2-30wk and estimated progress free survival (PFS) rate of 16 wk was 87 %. Standard and extend regimen were not different in effect, as well as the separated or concentrated infusion of rituximab (P>0.05). The regimen could be well tolerated, and the major adverse reactions were infusion-related response (32 % ) and hematological toxicities (20 %). CONCLUSION:Rituximab in combined with CHOP can be successfully applied to the therapy of initially diagnosed diffuse large B cell lymphoma, with high CR rate and mild adverse reactions.

EFFECTS OF INTEGRIN ALPHAⅡb^(R995A) MUTATION ON RECEPTOR AFFINITY AND pp125 (FAK) PHOSPHORYLATION

Objective To investigate the role of cytoplasmic domain of integrin alphaⅡb in platelet signal transduction. Methods Binding capacity of integrin alphaⅡb R995A to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting. Results Without activation, wild-type alphaⅡbbeta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alphaⅡb R995A beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alphaⅡbbeta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions. Conclusion The mutation in integrin alphaⅡb R995A alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.

IgE Myeloma： the First Report of Chinese Case and a Review of the Literature

To report a case of IgE myeloma and to compare its clinical features with those reported in the literature. Methods: M-component in serum and urine was determined using celluloseacetate membrane electrophoresis, immunofixation electrophoresis and quantification of immunoglobulins. Immunohistochemistry was performed to detect the expression of IgE and its light chain. Results: A monoclonal peak was detected by cellulose-acetate membrane electrophoresis. The monoclonal band showed on immunofixation electrophoresis the following reactive patterns: positive for A antisera and negative for κ, IgG, IgA, IgM and IgD antisera. The serum immunoglobulin concentrations were: IgG 21.6 g/L, IgA1.2 g/L, IgM 2.64 g/L, κ 7.49 g/L, λ 16.0 g/L, κ/A 0.47. The results of immunohistochemistry showed cytoplasmic expression of IgE immunoglobulin and A light chain in the tumor tissue. Conclusion: This is the first case of IgE multiple myeloma reported in China.

EFFECTS OF INTERLEUKIN-4 ON GRANULOCYTE-MACROPHAGE-COLONY FORMATION FROM MURINE BONE MARROW CELLS AND HEMATOPOIETIC RECONSTITUTION FOLLOWING MURINE ALLOGENEIC BONE MARROW TRANSPLANTATION

We investigated the effects of mouse recombinant IL-4 on hematopoiesis in vitro and in vivo. IL-4 alone was found to be incapable of stimulating colony formation, but it inhibited both IL-3-and GM-CSF-induced colony formation by murine hematopoietic progenitor cells. In contrast, colony formation induced by G-CSF was enhanced in the presence of IL-4. We also studied the influence of IL-4 on hematopoietic reconstiution after allogeneic bone marrow transplantation in a murine medel, and found that IL-4 had significant inhibitory effects on neutrophil recovery and that neutrophil recovery accelerated by IL-3 and G-CSF was significantly suppressed by IL-4. The combination of IL-4 and GM-SF caused a significant decrease in the absolute number of neutrophils.

Two-Dimensional （Polyacrylamide） Gel Electrophoresis Analysis of Apoptosis Induced by Harringtonine in K562 Cells

OBJECTIVE The anti-tumor drug, harringtonine (HT), has been extensively used with satisfactory results in the treatment of acute or chronic myeloid leukemia. Previous studies have shown that the anti-tumor activity of the drug is related to induced apoptosis of tumor cells, but the molecular mechanism still remains unclear. The main purpose of this research was to analyze the protein profiles formed during HT-induced apoptosis in K562 cells and to screen the apoptotic-related proteins. METHODS Annexin V and PI double staining was used in combination with flow cytometry to examine the early and the late stages of HT-induced apoptosis in K562 cells. In addition two-dimensional gel electrophoresis and computer-assisted image analysis were employed to separate and compare the HT-induced apoptotic proteins of the K562 cells and the controls. RESULTS When a concentration of 10 μg/ml HT was used to treat K562 cells, the percentage of the early-apoptotic cells (Annexin V+/PI-) was found to be 28.3% and 18.1% at 5 and 24 h, respectively (P<0.01), while the rate of lateapoptotic cells (Annexin V-/PI +) was at a level of 9.1% and 20.2% , respectively (P<0.01). Matching analysis of the proteome among the control group and the early- and late-apoptotic groups showed 1,300 ± 50 protein spots which were identified in the control K562 cells with a matching rate of 88.3 ± 2.0 % for the protein spots in the two treated groups. Ten protein spots showed overt and steady changes in both quality and quantity in the cells of the late-apoptotic group (P<0.01), among which the level of expression for eight of the ten protein spots was up-regulated after apoptosis, one was down-regulated and one was merely expressed as in the control cells. CONCLUSION The proteins with differential expression might be important proteins involved in the process of apoptosis in K562 cells induced by HT.

The Synergistic Inhibitory Effect of STI571 in Combination with Arsenic Trioxide （As2O3） on Multidrug-Resistant Leukemic Cells

OBJECTIVE To study the synergistic effect of STI571, an inhibitor of tyrosine kinase, in combination with arsenic trioxide As2O3 on a multidrug-resistant leukemia cell line expressing bcr-abl. METNODS The cytotoxic effect of STI571 alone or in combination with different concentrations of As2O3 on the bcr-abl and mdrl -positive leukemia cell line, K562-n/VCR, was examined by the MTT method. RESULTS One μmol/L of STI571 alone had no significant cytotoxic effect on K562-n/VCR cells. However the cytotoxic effect increased markedly when combined with As2O3 at concentrations of 10^-5, 10^-6, 10^-7 and 10^-8 mol/L. The IC50 of K562-n/VCR cells in As2O3 group was 1.879 μmol/L, with. Upon addition of STI571, the IC50 decreased to 0.155 μmol/L resulting in a synergistic cytotoxic effect on K562-n/VCR cells that was increased 12.1 times. CONCLUSION A combination of STI571 with As2O3 has a more powerful inhibitory effect on leukemia cells expressing positive bcr-abl and positive mdrl compared to the effect with As2O3 alone.